Equipment and methods for selecting, observing and separating sub-populations of biological cells, e.g., those contained in the blood, are known. Several methods which are considered to be important and indicative of the state of the art will now be described and commented on.
Separation based on cell adhesion. This method is not very efficient and is not suited for separation of different cells which have the same membrane characteristics, e.g., between cells that stick to glass.
Immunofluorescence separation. This method is based on known binding characteristics of sub-populations of cells to certain antigens and/or antibodies. The selective binding of these particular types of cells allows sub-populations of cells to be identified. However, this method is quite limited because it cannot distinguish subgroups within a group of cells based on specific biological activities and functions.
Electrophoresis. This method achieves cell-separation based on the relative rates of movement of different types of cells in an electric field. Thus, the method cannot be used to separate different groups of cells which respond to an electric field in the same manner.
Radio assay including radio immunoassay, radio incorporation assay, radio enzymological assay. In this method one cannot separate groups of cells from one another, nor distinguish a sub-group within a group based on specific biological activities and functions.
Morphology. Distinction between cells is based on their physical appearance. This method is quick but the coarsest of all.
Cell separation according to specific density (gradient technique). In this method cells float upon an isotonic solution of known density, osmolarity and viscosity. This configuration is subjected to acceleration forces by centrifugation at a given temperature and acceleration. The cells, having a specific weight greater than that of the solution sink. Those having the specific density of the solution are suspended in it, and those with a specific density less than that of the solution float above it. The main problem with this method is the cells' compartmentalization within the density gradient, which is influenced by ambient conditions such as temperature, osmolality, acceleration, e.g., the distance of the interface between the blood and the gradient from the spinning axis.
In addition to the above-stated shortcomings of the various prior art methods, a disadvantage common to all of them is due to the fact that the separated cells nearly always include cells belonging to other than the group or subgroup of interest. Therefore, the diagnosis which is based on the cell of interest is necessarily coarse, even if all the procedures have been carried out with the utmost precision.
For example, L. Cercek et al describe a SCM-test (Structuredness of Cytoplastic Matrix) in Biophys. J., July 1978, Vol. 23, No. 1. p. 395 ff. In said article the authors admit that by the above described gradient method, separated cells contain about 50% of undesired cells, in spite of the great care with which the test was conducted.
The inability to totally separate a particular group of cells from all others greatly affects diagnosis accuracy. Furthermore, and most significantly, in the above described methods, cell separation and the subsequent tests conducted thereon are on a macro or batch basis, rather than on a micro basis, i.e. one in which the selected cells are separated from one another and each cell can be separately tested and examined. Any system and method for separating selected cells of interest from other cells and further separating selected cells from one another, so that each can be separately tested and/or examined, would be of great significance in diagnosing various biological conditions and for other purposes. Testing and examining individual selected cells would eliminate errors, presently existing in many diagnoses, based on inexact statistical criteria.
A major object of the invention of U.S. Pat. No. 4,729,949 is to provide a method and equipment for selecting a group of cells from other cells and further separate the selected cells from one another. Each of the selected cells, separated from one another, is at a precisely known location. All of the selected cells are subjectable to common tests, yet the effect on each individual cell is determinable, thereby enabling more accurate diagnosis. The tests and the effects on each cell are performed automatically in order to reduce the testing time and to permit the task to be performed by relatively unskilled personnel.
Thus, U.S. Pat. No. 4,729,949 discloses and claims a method for placing individual cells at identifiable addresses within the holes of a carrier, and for performing on a cell-by-cell basis one or more of the operations of (i) observing or measuring a property of a living cell, (ii) moving a living cell, or (iii) killing a living cell, comprising the steps of:
(a) providing a carrier defining first and second outer surfaces and comprising an ordered array of holes therethrough, the positions on the carrier of the holes being identifiable and the holes being sized to contain individual living cells therewithin that the holes have (i) a first cross section at the first outer surface of the carrier of such dimensions that the living cell can pass through the first cross section without suffering substantial damage, (ii) a second cross section at a level spaced from the first outer surface of such dimensions that the living cell cannot pass through the second cross section, and (iii) a height between the first outer surface and the level of the second cross section such that either the entire living cell or substantially the entire living cell is contained within the hole so that the living cell is not washed out of the hole by the passage of a fluid across the first outer surface of the carrier;
(b) applying a fluid containing living cells to the first outer surface of the carrier;
(c) applying a force to the living cells to move the living cells into the holes; and where desired
(d) performing on an individual, first living cell located at a first hole having a first position on the carrier one or more of the operations of (i) observing or measuring a property of the first living cell, (ii) moving the first living cell, or (iii) killing the first living cell; and
(e) performing on an individual, second living cell located at a second hole having a second position on the carrier one or more of the operations of (i) observing or measuring a property of the second living cell, (ii) moving the second living cell, or (iii) killing the second living cell.
U.S. Pat. No. 4,729,949 also discloses and claims an apparatus for selecting particular living cells from among other living cells and for performing on a cell-by-cell basis one or more of the operations of (i) observing or measuring a property of a living cell, (ii) moving a living cell, or (iii) killing a living cell, comprising: (a) a carrier as defined in paragraph (a), above; (b) means for applying a fluid containing living cells to the first outer surface of the carrier; (c) means for applying a force to the living cells to move the living cells into the holes; and (d) means for performing on an individual, living cell located in one of the holes in the carrier one or more of the operations of (i) observing or measuring a property of the living cell, (ii) moving the living cell, or (iii) killing the living cell; and where (d) constitutes observing or measuring means, the apparatus optionally includes (e) means associated with such means, for recording the addresses on the carrier of particular living cells based on the results of observing or measuring the one or more properties.